Antimicrobial susceptibility in Clostridioides difficile varies according to European region and isolate source

Abstract Objectives Clostridioides difficile epidemiology is evolving with country-associated emerging and resistant ribotypes (RT). Antimicrobial susceptibility testing of C. difficile isolated from clinical and animal samples collected across Europe in 2018 was performed to provide antimicrobial resistance data and according to C. difficile RTs and source. Methods Samples were cultured for C. difficile and isolates PCR ribotyped. Metronidazole, vancomycin, fidaxomicin, moxifloxacin, clindamycin, imipenem, tigecycline, linezolid, rifampicin and meropenem minimum inhibitory concentrations (MICs) for 280 clinical and 126 animal isolates were determined by Wilkins–Chalgren agar dilution. Results Fidaxomicin was the most active antimicrobial (all isolates geometric mean MIC = 0.03 mg/L) with no evidence of reduced susceptibility. Metronidazole MICs were elevated among RT027 (1.87 mg/L) and RT181 clinical isolates (1.03 mg/L). RT027 and RT181 had elevated geometric mean moxifloxacin MICs (14.49 mg/L, 16.88 mg/L); clindamycin (7.5 mg/L, 9.1 mg/L) and rifampicin (0.6 mg/L, 21.5 mg/L). Five isolates (RT002, RT010 and RT016) were metronidazole resistant (MIC = 8 mg/L) and 10 (RT027; RT198) had intermediate resistance (4 mg/L). Metronidazole MICs were not elevated in animal isolates. Increased geometric mean vancomycin MICs were observed among RT078, mostly isolated from animals, but there was no resistance (MIC ≥ 4 mg/L). Clinical and animal isolates of multiple RTs showed resistance to moxifloxacin and clindamycin. No resistance to imipenem or meropenem was observed. Conclusion Increased antimicrobial resistance was detected in eastern Europe and mostly associated with RT027 and related emerging RT181, while clinical isolates from northern and western Europe had the lowest general levels of resistance.


Introduction
Antimicrobial resistance in C. difficile is a growing concern.As a colonizer of the gastro-intestinal tract of humans and animals, C. difficile may be exposed to selection pressures from multiple or sequential antimicrobial courses.Previous studies have identified multiple antimicrobial resistance markers in C. difficile PCR ribotypes (RTs), such as epidemic RT027 and highlighted emerging antimicrobial resistant RTs within specific geographical locations. 13][4] The Combatting Bacterial Resistance in Europe-Clostridioides difficile infections (COMBACTE-CDI) project is an IMI2 Horizon2020 framework project that aimed to develop a detailed understanding of the epidemiology and clinical impact of CDI across Europe. 5This afforded an excellent opportunity to examine the epidemiology of human and animal derived strains that were collected as part of this study to provide detailed and recent antimicrobial resistance data. 6The present study describes the results of phenotypic antimicrobial susceptibility testing.The antimicrobial testing panel was selected to provide data that can be compared with previous baseline data on anti-C.difficile agents (vancomycin, metronidazole, fidaxomicin) and those with known resistance in C. difficile (clindamycin, moxifloxacin, imipenem, rifampicin), while including other relevant (linezolid, meropenem and tigecycline) antimicrobials. 1

Ethics
The COMBACTE-CDI study is registered under the ClinicalTrials.govIdentifier NCT03503474.Ethical approval was received from participating countries and from the University of Leeds for the overarching study (IRAS244784).The planning conduct and reporting of this study was in line with the 2013 Declaration of Helsinki.Informed consent was not required for the use of anonymized residual diagnostic material and data. 6

Material
The collection of human clinical samples was previously described. 6riefly, all residual diarrhoeal faecal samples (from community and hospital patients) sent to 119 recruited testing facilities from 12 European countries, on two sampling days, during 2018 were collected, culture and PCR ribotyped. 6Contemporaneous C. difficile isolates were collected during the same period from animal faecal samples (mainly pigs and rodents) in similar European regions through the COMBACTE-CDI network practising veterinarians or authors of veterinary C. difficile publications.Participating countries represented European regions according to the United Nations Geoscheme as follows: the north: UK, Ireland and Sweden; the south: Greece, Italy and Spain; the east: Poland, Romania and Slovakia, and the west: Belgium (replaced by Austria for animal isolates), France and the Netherlands.Collected animal strains or samples were shipped to National Laboratory for Health, Environment and Food (NLZOH).C. difficile was isolated from animal faecal samples following enrichment for 5 days in brain heart infusion broth supplemented with 0.1% L-cysteine, 0.5% yeast extract, 0.1% taurocholic acid and C. difficile selective supplement (Oxoid, Basingstoke, UK), ethanol shock and cultured on selective medium (CHROMID C. Difficile, CHROMID ® C. difficile, bioMérieux, Marcy-l'Étoile, France).Before shipping animal C. difficile isolates from NLZOH to the University of Leeds for further testing, MALDI-TOF Biotyper System (Bruker, Bremen, Germany) was used to confirm C. difficile identification and crude agarose PCR ribotyping performed. 7ll samples received at the University of Leeds were cultured for C. difficile on CCEY agar (E and O laboratories, Scotland) and isolates were subsequently typed by PCR ribotyping following the C. difficile Ribotyping Network of England and Northern Ireland protocol (CDRN, Leeds, UK). 8,9

Antimicrobial susceptibility testing
Metronidazole, vancomycin, fidaxomicin, moxifloxacin, clindamycin, imipenem, tigecycline, linezolid, meropenem and rifampicin minimum inhibitory concentrations (MICs) for 280 clinical and 126 animal isolates were determined by Wilkins-Chalgren agar dilution as previously described. 1Briefly, antimicrobial dilutions were prepared according to solvents and diluents listed in the CLSI guidelines. 10C. difficile isolates were cultured anaerobically at 37°C for 24 h in pre-reduced Schaedlers anaerobic broths before adjustment to McFarland 1.0 equivalence and multipoint inoculation onto antimicrobial-containing agar plates.Inoculated plates were incubated for 48 h at 37°C in an anaerobic environment before examination.MICs were defined as the lowest concentration (in mg/L) of an antimicrobial that inhibited the growth of C. difficile.
Control strains were C. difficile NCTC 700057, B. fragilis NCTC and C. difficile E4 (PCR RT 010).Data are presented as the geometric means of MICs for each antimicrobial for all isolates, human clinical, animal isolates, per European regions and for the most common RTs (represented by at least five isolates).MICs for each antimicrobial and for each of the isolates are also available in Table S1 (available as Supplementary data at JAC-AMR Online).
MICs for each antimicrobial were scored as sensitive = 0, intermediate = 1 or resistant = 2 for each isolate according to published breakpoints used in the The ClosER study, and added to generate a cumulative resistance score (CRS) for each isolate. 1,11,12

Results
Two hundred and eighty clinical human isolates (81 distinct RTs) and 126 animal isolates (30 distinct RTs) were collected.RT distribution differed considerably between clinical and animal sources.RT181 was the most represented isolated clinical RT (9% of isolates), followed by RT027 (8%) and RT014 (6%).By contrast, among animal isolates RT078 accounted for over 32% of isolates, followed by RT126 (14%) and RT005 (8%).RT078 and highly related RT126 were the only RTs present in numbers >10 in both clinical and animal collections.
Fidaxomicin was the most active treatment agent (geometric mean for both clinical and animal isolates: geometric mean MIC = 0.03 mg/L, Table 1) with no evidence of reduced susceptibility among the isolates in this collection (Table S1).
There were some small differences in geometric mean MIC between clinical and animal isolates (Table 1).While no instances of vancomycin resistance were recorded among all tested isolates, geometric mean vancomycin MICs were marginally higher in animal versus clinical isolates (0.71 versus 0.52 mg/L, respectively).Isolates belonging to RT078 showed a slightly higher geometric mean vancomycin MICs in animal isolates compared to human clinical isolates (0.78 versus 0.69 mg/L, Tables 2 and 3).RT078 was more commonly isolated from animals than humans (41 versus 15, respectively), but there was no vancomycin resistance (MIC ≥ 4 mg/L, Table S1).
Average (mean) and median CRS showed that resistance levels among clinical (but not animal) isolates were highest in eastern Europe (Figure 2).

Discussion
There is increasing interest in the One Health approach to healthcare.The WHO particularly highlight the One Health approach as a potentially highly effective tool in combatting the spread of antimicrobial resistance. 13C. difficile is an organism that can be present in the gastro-intestinal tracts of humans and animals and in the environment, and as such is a prime example of the need for a One Health approach to antimicrobial resistance. 14he emergence of epidemic C. difficile RT027 in the early 2000s was notably associated with resistance to fluoroquinolones and, more recently, high-level resistance to cephalosporins has been also associated with this outbreak.The distribution of RTs among human clinical and animal isolates collected differed substantially in this study, with RT078 predominating in animals (>32% of isolates) and RT181 in human clinical isolates.The relatively high proportion of RT181 was influenced by the emergence of this RT in eastern Europe. 6Similarly high levels of emerging RTs (RT176 and RT198) were previously described in eastern European countries (Czech Republic and Hungary, respectively in a previous pan-European study of antimicrobial resistance). 1,17In both these cases, the emergent RTs also showed increased levels of antimicrobial resistance and were highly related to RT027, which was also prevalent in eastern Europe. 1 RT176 has also been described as the predominant strain in Slovakia in 2018-2019. 18In the present study, most clinical isolates for RT027 (76%; 16/21) also came from eastern Europe.This indicates the continuing predominance of this RT in this region and the emergence of closely related RTs in particular locations within it.It is worth noting that even though isolates in our study were collected in 2018, these represent the most recent information at the European level as there is limited European-wide data on RT distribution and corresponding antimicrobial resistance testing available for more recent years.Indeed, the most recent CDI annual epidemiological report from the European Centre for Disease Prevention and Control for the period 2018-2020 describes RT data beyond 2018 from only two countries, in part due to lower surveillance reporting during the SARS-CoV-2 pandemic. 19Therefore, meaningful comparison of the data presented hereby with more recent years is not yet possible.Interestingly, two recent national studies conducted between 2016 and 2019 identified RT181 as the predominant strain in Greece and RT027 as the second most prevalent type in Denmark. 20,21This study presented the opportunity to examine and compare antimicrobial resistance of human and animal isolates from the same locations [albeit in smaller numbers (clinical versus animal isolates: east: 80 versus 25; north: 83 versus 3; south 82 versus 31; west 35 versus 67, average: 70 clinical versus 32 animal isolates)].RT181 and RT027 isolates were, however, not identified in this animal collection, in accordance with previous studies. 22RT078 was the only RT present in both human clinical and animal collection in numbers >10 isolates, highlighting the association of this RT with both humans and animals. 2,3esistance to antibiotics used in the treatment of CDI among both clinical and animal isolates was reassuringly low, with 15 clinical isolates demonstrating metronidazole MICs above the European Committee on Antimicrobial Susceptibility Testing breakpoint (>2 mg/L, 11 ), but only five with MICs >4 mg/L, and no instances of vancomycin or fidaxomicin resistance.While geometric mean vancomycin MICs in animal derived RT078 isolates were slightly higher (0.78 mg/L), none displayed vancomycin resistance, which is uncommon among C. difficile. 1owever, there was evidence of reduced susceptibility to metronidazole among RT027 and RT181 isolates from eastern Europe (GM MICs 1.87 and 1.03 mg/L, respectively).The mechanisms behind metronidazole resistance are only recently beginning to be understood, and the reasons for their emergence remain unclear. 23,246][27] Surveillance of C. difficile RTs with this phenotype is therefore important, particularly in the light of poor gut metronidazole levels. 28Geometric MICs for clinical isolates were reflective of those observed in the large pan-European study by Freeman et al. and geometric mean MICs for animal isolates were similar. 1 However, when general levels of resistance (to all antimicrobials) were assessed according to a CRS, these were higher in clinical isolates, and in particular those from eastern Europe.This is probably driven by RT027 and the emergent antibiotic resistant RT181 in Romania: together accounting for accounting for 51.3% (41/80) of human clinical isolates submitted from eastern Europe.While there were moxifloxacin and clindamycin resistant isolates in both clinical and animal collections, neither exhibited high-level resistance (>128 mg/L) such as that previously reported in epidemic human-associated RTs such as RT027 and 001, 1 but high-level resistance to rifampicin was common among RT027 and RT181.Consumption rates of rifampicin and quinolones in Romania were among the highest submitted to ESAC-net in 2018. 29While this is in no way definitive evidence, it is possible that multiple antimicrobial selectors may contribute to the persistence and emergence of RTs in particular locations.There was much less evidence of multiple antimicrobial resistance among animal isolates; clindamycin resistance was mainly observed among RT005, RT014 and RT045 (GM mean MICs 14.93, 12.13 and 51.98 mg/L, respectively) and notably with RT045 animal isolates associated with eastern Europe (9/ 10).Interestingly, geometric mean clindamycin MICs were lower in clinical RT005 and RT014 isolates (6.56 and 8.00 mg/L, respectively).However, this is probably a reflection of origin: animal RT005 and RT014 were associated with western Europe (8/10 and 4/5, respectively), while clinical RT005 and RT014 were isolated in all four European regions.Only a single clinical RT045 was isolated, from eastern Europe, and this was clindamycin resistant.No antimicrobial resistance was detected in isolates of RT695, which was only identified in our animal collection from western Europe (five isolates from the Netherlands).Similarly, RT695 emerged as the most prevalent type in cattle from Dutch dairy farms in 2021. 30his study highlights the continued prevalence of antimicrobial resistant epidemic clinical C. difficile RT isolates, along with the emergence of closely related and similarly resistant RTs in particular geographic locations.While this may be linked to local antimicrobial prescribing policies, robust epidemiological and clinical studies are needed.Analysis of genome sequence data and continued surveillance will further enhance our knowledge of C. difficile resistance emergence and epidemiology.partners: Pfizer Ltd, GlaxoSmithKline, bioMérieux, Sanofi Pasteur and Da Volterra.The management board of COMBACTE-CDI consists of Marc Bonten (UMC Utrecht), Philippe Cleuziat (bioMérieux), Chris Webber (Pfizer Ltd), Maja Rupnik (NLZOH) and Mark Wilcox (University of Leeds).We would also like to acknowledge the staff of CDRN, led by Dr Warren Fawley, for PCR ribotyping.The authors would like to acknowledge the help of colleagues in collecting the animal samples and animal isolates: Kornelia Maccari-Hrastnig, Joanna Wojtacka, Jana Koscova, Emil Pilipcinec, Vlad Emil Luca, Jose Luis Blanco, Rosa Bolea, Magdalena Jacobson, Caroline Le Marechal, Guillaume Lequeux, Maarten Verstraeten, Celine Harmanus, Ed J. Kuijper, Daniela Pasotto and Illenia Drigo.

Funding
COMBACTE-CDI received support from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement number 777362, resources of which are composed of contributions from the European Union's Horizon 2020 research and innovation programme, and European Federation of Pharmaceutical Industries and Associations.The funder contributed financially to study design but had no role in the collection, analyses, or interpretation of data, in the writing of the manuscript; or in the decision to publish the results.

Figure 2 .
Figure 2. Antimicrobial resistance in clinical human and animal C. difficile isolates by European region.Average CRS is shown (median as black line).
Resistance breakpoints used for CRS calculation were as follows: MICs ≥ 8 mg/L for metronidazole, vancomycin, moxifloxacin and clindamycin (intermediate score if ≥4 mg/L); MICs ≥16 mg/L for imipenem and meropenem (intermediate score if ≥8 mg/L); MICs >4 mg/L for linezolid; MICs >1 mg/L for fidaxomicin; MICs >0.25 mg/L for tigecycline and MICs ≥16 mg/L for rifampicin (intermediate score if 0.008-16 mg/L).The average (and median) CRSs across all human or animal isolates and per European region are presented.

Table 1 .
Geometric mean MICs for all antibiotics tested against human clinical (n = 280) and animal C. difficile isolates (n = 126)